Alzheimer's disease (AD) is the most common form of dementia in the elderly. It is characterized by two hallmark lesions: senile plaques (SPs) and neurofibrillary tangles (NFTs). Sps, an early and invariant feature of AD, are composed largely of the beta-amyloid peptide (betaA4). The BetaA4 peptide sequence is found within the amyloid precursor protein (APP) from which it is liberated by posttranslational proteolytic processing. While some patient with familial AD have mutations within the APP gene, most do not. Rather, it increasingly appears that altered APP processing in neurons is responsible for BA4 generation, and that this plays a critical role in AD pathogenesis. Determining how BetaA4 is generated from APP has been complicated by the fact that APP processing is strongly cell type dependent and by the lack of an adequate in vitro neuronal model system. A recently developed method to obtain >99% pure, fully differentiated postmitotic human CNS neurons provides the means to study APP processing in vitro in neurons. Preliminary studies show that these cells (NT2N cells) express a major CNS APP isoform (APP695) and process it to a unique array of C-terminal amyloidogenic fragments. Of particular importance is that these cells produce, and secrete into the medium, intact BetaA4. This grant proposal takes a cell biological approach to study the biosynthesis and processing of APP in NT2N cells. Important issues to be addressed include the role of axonal transport in APP processing and determining the site(s) of APP cleavage and BetaA4 generation. Close collaborations with other investigators in this Program Project will make it possible to study the roles of several important posttranslational processes, such as glycosylation and phosphorylation, in APP biosynthesis and processing. The completion of the studies proposed here will provide important information on how the BetaA4 peptide is generated in neurons.